The extraction of Scutellaria baicalensis Georgi was investigated using the response surface methodology-genetic algorithm mathematical regression model, and the extraction variables were optimized to maximize the flavonoid yield. Furthermore, a simple and efficient ultrafiltration-liquid chromatography-mass spectrometry and molecular docking methods were developed for the rapid screening and identification of acetylcholinesterase inhibitors present in Scutellaria baicalensis Georgi. Subsequently, four major chemical constituents, namely baicalein, norwogonin, wogonin, and oroxylin A, were identified as potent acetylcholinesterase inhibitors. This novel approach, involving the use of ultrafiltration-liquid chromatography-mass spectrometry and molecular docking methods combined with stepwise flow rate counter-current chromatography and semi-preparative high-performance liquid chromatography, could potentially provide a powerful tool for the screening and extraction of acetylcholinesterase inhibitors from complex matrices and be a useful platform for the production of bioactive and nutraceutical ingredients.

Introduction Medicago sativa contains flavonoids, saponins, coumarins, sterols, monoterpenes, and organic acids, with flavonoids being the main active constituents. Flavonoids naturally contain a 2‐phenylchromone structure with antioxidant, free radical scavenging, cardiovascular, and trace estrogen‐like effects. Objective Screening and isolation of neuraminidase, lipoxidase, and lactate dehydrogenase inhibitors from M. sativa via ultrafiltration‐liquid chromatography‐mass spectrometry (UF‐LC–MS) combined with stepwise flow rate counter‐current chromatography (CCC). Method Utilising the medicinal plants M. sativa as the research objects and UF‐LC–MS was used for activity screening followed by isolation and purification of the inhibitors by stepwise flow rate CCC. Finally, identification of the three active compounds was achieved by MS and nuclear magnetic resonance. Results Three major compounds, viz. quercetin, genistein, and formononetin, were identified as potent neuraminidase, lipoxidase, and lactate dehydrogenase inhibitors, respectively. A two‐phase solvent system of ethyl acetate/methanol/n‐butanol/water (5.0:1.5:5.0:10; v/v/v/v) was subsequently selected for separation by stepwise flow rate CCC. Conclusion This novel approach based on UF‐LC–MS and stepwise flow rate CCC represents a powerful tool for the screening and isolation of neuraminidase, lipoxidase, and lactate dehydrogenase inhibitors from complex matrices. Therefore, a useful platform for the large‐scale production of bioactive and nutraceutical ingredients was developed herein.

We present a simple and efficient method based on ultrafiltration high‐performance liquid chromatography coupled with a photodiode array detector and electrospray ionization mass spectrometry for the rapid screening and identification of ligands obtainable from the extract of Scutellaria baicalensis. Five major compounds (chrysin‐6‐C‐arabinosyl‐8‐C‐glucoside, chrysin‐6‐C‐glucosyl‐8‐C‐arabinoside, baicalin, oroxylin A‐7‐O‐glucuronide, and wogonoside) were identified as potentially effective inhibitors of lipoxidase and superoxide dismutase. Subsequently, specific binding ligands were separated by high‐speed countercurrent chromatography, using ethyl acetate/ethyl alcohol/water acetate (0.1%) (1.0:0.1:1.0, v/v/v) as the solvent system. To the best of our knowledge, this is the first report of S. baicalensis extracts containing potent lipoxidase and superoxide dismutase inhibitors. Our results demonstrate that the systematic isolation of bioactive components from the n‐butyl alcohol layer of S. baicalensis guided by ultrafiltration high‐performance liquid chromatography coupled with photodiode array detection and electrospray ionization mass spectrometry represents a feasible and efficient technique that could also be employed for the identification and isolation of other enzyme inhibitors.

A simple and efficient method was developed for the rapid screening and identification of ligands for monoamine oxidase B. A new ionic‐liquid‐based ultrasound‐assisted extraction method for medicinal herbs was also developed and validated. In addition, the hyphenated technique of countercurrent chromatography and semipreparative‐LC was developed and applied to the isolation of the chemical constituents for Pueraria thomsonii Benth. Three potent monoamine oxidase B inhibitors, namely, daidzein‐4′,7‐diglucoside (42.2 mg), puerarin 6′′‐O‐xyloside (88.3 mg), and 3′‐hydroxypuerarin (48.5 mg) with purities of 98.2, 96.3, and 97.1%, respectively, were obtained from 500 g of P. thomsonii raw material using semi‐preparative high‐performance liquid chromatography, whereas 3′‐methoxypuerarin (76.2 mg), daidzein‐8‐C‐apiosyl (1→6) glucoside (84.2 mg), and tectorigenin (75.1 mg) with purities of 98.5, 96.4, and 96.8%, respectively, were obtained from 500 g raw material via countercurrent chromatography using a two‐phase solvent system comprising n‐hexane–ethyl acetate–methanol–water at a volume ratio of 1.85:1.00:0.86:3.69 (v/v/v/v). Then, the anti‐Alzheimer activity of the phytochemicals was assessed using a PC12 cell model. Treatment with tectorigenin, daidzein‐4′,7‐diglucoside, puerarin 6′′‐O‐xyloside, 3′‐hydroxypuerarin, 3′‐methoxypuerarin, and daidzein‐8‐C‐apiosyl (1→6) glucoside (100 μg/mL), resulted in cell viabilities of 69.00, 65.81, 59.69, 57.90, 55.61, and 54.59%, respectively (p < 0.001). The protocol was proved to be very accurate and efficient.

Development of a method to screen and isolate xanthine oxidase inhibitors from black bean in a single step: hyphenation of semipreparative liquid chromatography and stepwise flow rate countercurrent chromatography.